Method for preparing 2,5-diketo-D-gluconic acid

ABSTRACT

2,5-Diketo-D-gluconic acid is prepared in high yield and in high broth concentration by cultivating newly isolated microorganisms of genus Erwinia in an aqueous nutrient medium in the presence of D-glucose. The production is also possibly by simple contact of said microorganisms or their processed products therefrom, with D-glucose.

This application is a Divisional of application Ser. No. 07/403,298 filed on Sep. 5,1989, now U.S. Pat. No. 5,134,077 which is a divisional of application Ser. no. 07/243,215 filed on Sep. 12, 1988, now U.S. Pat. No. 4,879,229 which is a continuation of application Ser. No. 06/925,538 filed on Oct. 28, 1986, now abandoned, which is a continuation of application Ser. No. 06/288, 332 filed on Jul. 30, 1981, now abandoned, each expressly incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel microbiological method for preparing 2,5-dike to-D-gluconic acid (hereinafter referred to as 2,5 DKG) from D-glucose. Particularly, it is concerned with a method for preparing 2,5-DKG by fermentation of new microorganisms which belong to Erwinia n. sps. or by simple contact of D-glucose with said microorganisms or their processed products, for instance, resting cells, immobilized cells, ground cells or any enzymes extracted therefrom, which may be suspended in a fluid medium or fixed on a surface of a stationary bed.

2. Description of the Prior Art

2,5 DKG is a useful intermediate in the production of L-ascorbic acid. Namely, 2,5 DKG can selectively and stereospecifically be reduced into 2- keto-L-gulonic acid, a precursor of L-ascorbic acid, in a promising new route for the latter product (See, for instance, U.S. Pat. No. 3,922,194 3,959,076, 3,963,574 and 3,998,697).

In all of the known means, the production of 2,5 DKG has been achieved by cultivating aerobic microorganism strains which belong exclusively to genera Acetobacter, Acetomonas, Gluconobacter and Pseudomonas. No instance has hitherto been reported as to its production by means of cultivation using faculatatively anaerobic strains which belong to genus Erwinia.

SUMMARY OF THE INVENTION

It is therefore a primary object of the present invention to provide a method for preparing 2,5 DKG by the use of newly isolated microorganisms which belong to genus Erwinia.

It is still another object of the present invention to provide a method for preparing 2,5 DKG in a high yield and in a high broth concentration to facilitate a commercialization of the previously mentioned promising new route.

According to the present invention, there is provided a method for preparing 2,5 DKG characterized by contacting a 2,5 DKG producing microorganism of genus Erwinia or any processed products therefrom (any product obtained by treating cells of said microorganisms) with D-glucose.

The present invention may be embodied in a variety of modes, for instance, by cultivating at least one strain of said microorganisms in an aqueous nutrient medium containing D-glucose or by simply contacting said microorganisms or their processed products, i.e., resting cells, immobilized cells, or cell free enzymes extracted therefrom, with any substrate containing D-glucose.

Throughout this specification and claims, the term "cultivating" is used to include any modes of microbiological operations wherein the microorganisms are inoculated into and incubated in a medium to effect a fermentative action therein. The term "contacting" is used to mean operations wherein the microorganisms or any processed products therefrom are combined with the substrate to effect a conversion of D-glucose into 2,5 DKG, without regard to the length of the contact, i.e., incubation.

In the process of the present invention, any microorganisms of genus Erwinia can be utilized as far as the microorganisms are capable of selectively oxidizing D-glucose and converting it into 2,5 DKG. Examples of these strains, which have been isolated and determined to belong to new species of genus Erwinia by the present inventors, are collectively listed in Table 1 below.

All of the listed strains, which have been deposited with the Fermentation Research Institute, Japan and with the American Type Culture Collection, Washington D.C., to be assigned FERM-P and ATCC numbers, respectively, are found to below to the three new species and each constitutes variant with respect to the other within the species.

                  TABLE 1                                                          ______________________________________                                                 Name given to the isolated                                             SHS-    microorganism     FERM-P   ATCC                                        ______________________________________                                         2003    Erwinia citreus   5449     31623                                       2006    Erwinia punctata  5452     31626                                       2004    Erwinia punctata var.                                                                            5450     31624                                       2005    Erwinia punctata var.                                                                            5451     31625                                       2007    Erwinia punctata var.                                                                            5453     31627                                       2008    Erwinia terreus   5454     31628                                       2009    Erwinia terreus var.                                                                             5455     31629                                       2010    Erwinia terreus var.                                                                             5456     31630                                       2011    Erwinia terreus var.                                                                             5457     31631                                       ______________________________________                                    

Details of taxonomic studies on these microorganisms will be collectively described in Table 2, below.

Table 2 A. Observations 1. Shape of cells

(Bouillon agar slants at 28° C. for 24, 72 and 168 hrs.) Cells predominantly single, rods of the listed dimensions with rounded ends in common with each of the observed strains. Sometimes, the presence of deformed cells of the indicated dimensions are observed in some strains.

    ______________________________________                                                       Deformed cells:                                                  Strain:                                                                              Predominant cells:                                                                           Expand-  Extended cells:                                   SHS-  (Dimensions (μ))                                                                          ed cells:                                                                               (Dimensions (μ))                               ______________________________________                                         2003  0.8-1.2 × 1.0-2.9                                                                      Observed Observed,                                                                                 1.6 ×                                                                 6.0-7.0                                   2004  1.0-1.5 × 1.5-3.3                                                                      "        "       0.7-0.8 ×                                                                4.0-5.0                                   2005  1.0-1.3 × 1.6-2.1                                                                      "        "       0.7-0.8 ×                                                                2.4-3.0                                   2006  1.1-1.3 × 1.3-1.9                                                                      "        "       0.6-0.8 ×                                                                2.4-2.7                                   2007  1.0-1.2 × 1.7-2.3                                                                      None     None    --                                        2008  0.8-0.9 × 1.5-1.7                                                                      "        "       --                                        2009     1.0 × 1.2-2.1                                                                       "        "       --                                        2010     1.0 × 1.0-2.0                                                                       "        "       --                                        2011     1.0 × 1.3-2.0                                                                       "        "       --                                        ______________________________________                                    

2. Motility and flagellation

(Bouillon agar slants at 20° C. and 25° C. for 18 through 24 hrs.)

Motility is confirmed by the hanging-drop method. Flagellation is confirmed under an optical microscope after being stained by Toda's method, or by means of transmission type electron microscope after being cultured in a sustained membrane culturing method. Strains SHS-2003, 2004, 2005, 2006 and 2007: Non-motile in common.

Strain SHS-2008: Motile with mono-lateral flagellum.

Strains SHS-2009, 2010 and 2011: Motile with one or two lateral flagella.

3. Spore

Not formed with each of the listed strains in common.

4. Gram staining

(Bouillon agar slants at 28° C. for 24, 72 and 168 hrs.) Negative with each of the listed strains in common.

5. Acid fast

Negative in common.

B. Growth on a variety of media

1. Bouillon agar colonies at 28° C. for 24, 48, 72 and 168 hrs. Initial colonies being circular, entire, smooth, translucent and butyrous in common.

    ______________________________________                                         2004  "         Pale    Smooth but gradually changes to                                        beige   rough.                                                 2005  "         Pale    Convex but gradually changes to                                        beige   raised.                                                2006  "         Pale    Convex but gradually changes to                                        beige   umbonate.                                              2007  "         Pale    Raised but gradually changes to                                        beige   umbonate.                                              2008 Dull to    Pale    Smooth but gradually changes to                             glistening reddish rough and convex. Dull but grad-                                       yellow  ually changes to glistening.                           2009 Glistening Pale    Smooth but gradually changes to                                        beige   rough and raised.                                      2010  "         Pale    Convex but gradually changes to                                        beige   umbonate.                                              2011  "         Pale    Convex but gradually changes to                                        reddish flat. Viscid but gradually                                             yellow  changes to butyrous.                                   ______________________________________                                    

2. Bouillon agar slant at 28° C. for 24-168 hours. Initial colonies being filiform and butyrous in common.

    ______________________________________                                         Strains                         Progress of culture                            SHS-  Growth    Brilliancy                                                                               Color (change)                                       ______________________________________                                         2003  Abundant  Glistening                                                                               Pale  --                                                                       reddish                                                                        yellow                                               2004    "       Dull to   Pale  Dull but gradually                                             glistening                                                                               beige changes to glistening.                         2005  Moderate  Glistening                                                                               Pale  --                                                                       beige                                                2006    "       Dull to   Pale  Dull but gradually                                             glistening                                                                               reddish                                                                              changes to glistening.                                                   yellow                                               2007  Scanty    Dull      Pale  --                                                                       reddish                                                                        yellow                                               2008  Abundant  Dull to   Pale  Dull but gradually                                             glistening                                                                               reddish                                                                              changes to glistening.                                                   yellow                                               2009    "       Dull to   Pale  Dull but gradually                                             glistening                                                                               reddish                                                                              changes to glistening.                                                   yellow                                               2010  Moderate  Glistening                                                                               Pale  --                                                                       reddish                                                                        yellow                                               2011    "         "       Pale  --                                                                       reddish                                                                        yellow                                               ______________________________________                                         3. Buillon broth at 28° C. for 24-168 hours. No odor in common.         Growth on the surface                                                          of culture                                                                     Ring along  Membran-  Floc-   Submerged growth                                 Strains                                                                              the tube  ous       culent       Flocculent                              SHS-  wall      growth    growth                                                                               Turbidity                                                                             sediment                                ______________________________________                                         2003  On and    --        --    Slight Scanty                                        after 3rd                                                                      day                                                                      2004  --        On 6th day                                                                               --    Slight to                                                                             Initial                                                                 moderate                                                                              compact                                                                        sediment                                                                       changes                                                                        to scanty                                                                      flocculant                                                                     sediment                                2005  --          "       --    Slight Scanty                                  2006  --          "       --    Slight to                                                                              "                                                                      moderate                                       2007  On 3rd day                                                                               On 7th day                                                                               --    Slight  "                                      2008  On 7th day                                                                               --        On 3rd                                                                                "      "                                                                day                                                  2009  --        On 3rd to On 7th                                                                                "      "                                                      6th day   day                                                  2010  --        On 6th day                                                                               --    Slight to                                                                              "                                                                      moderate                                       2011  --        On 4th day                                                                               --    Slight  "                                      ______________________________________                                    

4. Bouillon gelatin stab at 20° C. for 40 days. No liquefaction in common. Growth along the stab line.

5. Litmus milk at 28° C. for 40 days.

strain SHS-2003: acidification beings within 7 days. Weak and uniform coagulation begins on about the 18th day and is completed on the 38th day. From the 18th day to the 32nd day, the upper-layer becomes pink and the lower-layer becomes grayish brown, but on the 38th day, whole layer becomes pink in uniform.

Strains SHS-2004, 2005, 2006, 2007, 2008, 2009, 2010 and 2011: No changes are observed throughout the incubation in common.

6. Potato slant at 28° C. for 24-168 hours.

filiform, butyrous and glistening in common.

    ______________________________________                                         Strains          Growth      Color                                             ______________________________________                                         SHS-2003         Abundant    Pale reddish yellow                               2004               "         Beige                                             2005             Moderate     "                                                2006               "         Pale beige                                        2007               "         Beige                                             2008             Abundant     "                                                2009               "          "                                                2010             Moderate     "                                                2011               "          "                                                ______________________________________                                    

C. Physiological properties (Unless othersiwe indicated, based on the results of the observation at 28° C. within 14 days)

1. Nitrite: Nitrite is produced from nitrate in common.

2. Nitrate respiration: Neither growth nor gas production is observed in paraffin-sealed bouillon broth containing 1% KNO₃ in common.

3. Methyl-red test: Positive in common, except strain SHS-2010 which is only very weakly positive.

4. Voges-Proskauer reaction (glucose):

Strain SHS-2003: Very weakly positive.

Strains SHS-2004, 2005, 2007, 2008, 2010 and 2011: Positive.

Strains SHS-2006 and 2009: Negative or very weakly positive.

5. Indole: Negative in common.

6. Hydrogen sulfide:

(i) Bacto peptone water-lead acetate paper: Positive in common.

(ii) TSI Agar: Negative in common.

(iii) Kliegler agar: Negative in common.

7. Ammonia: Not produced in common.

8. Hydrolysis of starch: Negative in common.

9. Growth on citrate media:

(i) Simmon's medium*: Growth in common.

(ii) Christensin's medium: Growth in common.

10. Growth with inorganic nitrogen sources:

(i) Ammonium (Glucose-Hucker's medium)*: Growth in common.

(ii) Nitrate (Glucose-Dimmick's medium)*: Growth is observed for Strains SHS-2003, 2004, 2005, 2008, 2009, 2010 and 2011: while SHS-2006 does not grow and SHS-2007 only grows very weakly.

(i) Blue pigment (Yeast extract 1%, glucose 1%, CaCO₃ 2%, at 28° C. for 7 days): Negative in common.

(ii) Pink diffusible pigment (the same medium as above): Negative in common.

(iii) Yellow pigment (bouillon agar at 28° C. for 7 days): Negative in common.

12. Urease: Negative in common.

13. Catalase: Positive in common.

14. Oxidase (bouillon agar slant, for 18-24 hours, containing tetramethylphenylenediamine): Negative in common.

15. Temperature relations (Bacto yeast extract 0.5%, Bacto peptone 0.5% and glucose 0.5%, pH 7):

    ______________________________________                                         Strains     Temp. of   Optimum temp. of                                        SHS-        growth (°C.)                                                                       growth (°C.)                                     ______________________________________                                         2003        8.5-38.5   18-31                                                   2004        6.5-40.0   20-34                                                   2005        6.5-38.5   22-28                                                   2006        6.5-38.5   21-28                                                   2007        4.0-47.5   21-27                                                   2008        18.0-40.0  19.5-32.5                                               2009        8.5-38.5   24-30                                                   2010        6.5-40.0   20-34                                                   2011        4.0-40.0   19.5-24                                                 ______________________________________                                    

16. pH relations (glycerol 1%, Bacto yeast extract 0.05%, Bacto peptone 1%, NaCl 0.5%, in 0.1 M 3,3-dimethylglutaric acid buffer, 28° C. for 2-4 days)

    ______________________________________                                         Strains                Optimum pH of                                           SHS-        pH of growth                                                                              growth                                                  ______________________________________                                         2003        5.5-8.0    6.0-7.5                                                 2004        5.5-8.5    6.0-7.5                                                 2005        5.5-8.5    6.0-7.5                                                 2006        5.5-8.5    6.0-7.5                                                 2007        5.5-8.5    6.0-7.5                                                 2008        5.5-7.5    6.0-7.5                                                 2009        5.5-8.0    6.0-7.5                                                 2010        6.0-8.0    6.0-7.5                                                 2011        5.5-7.5    6.0-7.5                                                 ______________________________________                                    

17. Oxygen requirement: Facultatively anaerobic in common.

(i) Liquid paraffin sealed stab culture:

(Glucose-BCP medium, containing Bacto beef extract 1%, Bacto peptone 1%, Bacto yeast extract 0.2%, NaCl 0.5%, bromocresol purple 0.004% and agar powder 1.5%, pH adjusted at 7.0-7.2, was dispensed in test tubes of 18 mm in diameter in a depth of about 8 cm. The dispensed medium was sterilized at 121° C. for 15 min. Stab inoculation was performed before complete solidification of the medium. The test tubes were then sealed with sterile liquid paraffin (5-7 ml) in 4 cm depth.) Tubes were cultured at 28° C. and observed over a period of 7 days.

There is observed a distinct growth along the stab line from the top to the bottom of the medium within 2-3 days and the culture begins to become slightly yellowish uniformly to show a distinct difference in color from the sealed cultured tube of the non-inoculated control with each of the listed strains. The degree of yellow color further increases with the lapse of time.

(ii) Gas-Pak anaerobic system (BBL):

(Bacto nutrient agar plate, containing Bacto beef extract 1%, Bacto peptone 1%, Nacl 0.5% and agar powder 1.5%, or GYP agar plate, containing glycerol 0.5%, Bacto yeast extract 0.5%, Bacto peptone 0.3%, KH₂ PO₄ 0.1%, MgSO₄.7H₂ O 0.02% and agar powder adjusted at 7.0-7.2), Sligh streak inoculation of a suspension of the test organism was cultured at 28° C. for 48 hrs. after the oxygen was removed by two hour's reaction at 45° C. in an incubator.

Only weak growth is observed in common. The growth is weaker than that of Escherichia coli used as control.

18. O-F test (Hugh-Leifson's method): Fermentative in common. Medium containing Bacto trypton 1%, Bacto yeast extract 0.1%, bromocresol purple 0.004%, glucose (or lactose) 1% and agar powder 0.2%, pH adjusted at 7.0-7.2 was dispensed to test tubes (18 mm in diameter) in a depth of about 8 cm. Dispensed medium was sterilized at 121° C. for 15 min. After the temperature of the medium became about 30°-40° C., duplicate inoculations were performed for each organism of the listed strains, and one tube of each pair was then sealed with sterile liquid paraffin (5-7 ml) about 4 cm in depth. Tubes were cultured at 28° C. and observed over a period of 7 days.

Acid is produced under both aerobic and anaerobic conditions from D-glucose (also from lactose, in the case of SHS-2003), but no gas. A distinct growth along the stab line from the top to the bottom is observed with the sealed culture in as the non-sealed culture. Anaerobical acid formation is not so rapid as Escherichia coli used as control

19. Production of acids and gases from carbohydrates:

Modified Barsikow's method at 28° C. for 7 days, stationary)

    __________________________________________________________________________     Strains                                                                            Fruc-                                                                              Glu-                                                                               Galac-                                                                              β-Methyl- Manni-           Meli-     Gly-                SHS-                                                                               tose                                                                               cose                                                                               tose glucoside                                                                            Mannose                                                                             Ribose                                                                             tol Salicin                                                                            Xylose                                                                             Lactose                                                                             biose                                                                              Cellobiose                                                                           cerol               __________________________________________________________________________     2003                                                                               +   +   +    +     +    +   +   +   +   +    +   +     +                   2004                                                                               +   +   +    +     +    +   -   +   +   -    +   -     +                   2005                                                                               +   +   +    +     +    +   -   +   +   -    +   +     +                   2006                                                                               +   +   +    +     +    +   -   +   +   -    +   +     -                   2007                                                                               +   +   +    +     +    +   -   +   +   -    +   +     -                   2008                                                                               +   +   +    +     +    +   -   +   +   -    +   +     +                   2009                                                                               +   +   +    +     +    -   -   +   +   -    +   +     +                   2010                                                                               +   +   +    +     +    ±                                                                               -   +   +   -    +   +     -                   2011                                                                               +   +   +    +     +    -   -   +   +   -    +   +     -                   __________________________________________________________________________     Strains                                                                            Su- Mal-                                                                               Dex-       Rham-                                                                               Sorbi-                                                                             Raffi-                                                                             Adoni-                                                                             Dulci-                                                                             Melezi-                                                                             Arabi-                                                                             α-Methyl-                                                                      Inosi-              SHS-                                                                               crose                                                                              tose                                                                               trin Esuclin                                                                              nose tol nose                                                                               tol tol tose nose                                                                               glucoside                                                                            tol                 __________________________________________________________________________     2003                                                                               -   -   -    -     -    -   -   -   -   -    -   -     -                   2004                                                                               +   -   -    -     -    -   -   -   -   -    -   -     -                   2005                                                                               +   -   -    -     -    -   ±                                                                               -   -   -    -   -     -                   2006                                                                               +   -   -    -     -    -   -   -   -   -    -   -     -                   2007                                                                               +   -   -    -     -    -   ±                                                                               -   -   -    -   -     -                   2008                                                                               +   -   -    -     -    -   -   -   -   -    -   -     -                   2009                                                                               +   -   -    -     -    -   -   -   -   -    -   -     -                   2010                                                                               +   -   -    -     -    -   -   -   -   -    -   -     -                   2011                                                                               +   -   -    -     -    -   -   -   -   -    -   -     -                   __________________________________________________________________________      Note:                                                                          (i) Acid but no gas, produced (+)                                              (ii) Slight acid but no gas, produced (±)                                   (iii) Neither acid nor gas, produced (-)                                 

20. Methylene blue (bouillon broth, 18-24 hrs.): Reduced in common.

21. D-Gluconic acid: Utilized in common.

22. 2-Keto-D-gluconic acid: Utilized in common.

23. Production of reduced compound from sucrose (Bergey's manual of determinative bacteriology 8th Ed. (1974) page 335, Table 8.19, foot note f): Positive in common except for SHS-2003.

24. Decarboxylation of various amino acids:

(Shaw, C & Clarke, P.H., J. Gen. Microbiol., 13, 155-161 (1955))

(i) L-glutamic acid: Negative with SHS-2003, 2008, 2009, 2010 and 2011, Positive with SHS-2004, 2005 2006, and 2007.

(ii) L-lysine: Negative in common.

(iii) L-arginine: Negative in common.

(iv) L-ornithine: Negative in common.

25. Lipase (Modified Starr's method, Starr, M.P., Science, 93, 333-334 (1941)): Negative in common.

26. D-gluconate oxidation (Shaw, C & Clarke, P.H., J. Gen. Microbiol., 13 155-161 (1955)): Positive in common.

27. Pectate degradation (A. M. Paton, Nature, 183, 1812-1813 (1959)): Negative in common.

28. Casein hydrolysis (D.W. Dye, N.Z. J. Sci., 11, 590-607 (1968)): Negative in common.

29. Symplasmata (Graham, D.C. & Hodgkiss, W., J. Appl. Bacteriol., 30(1), 175-189 (1967)): Negative with SHS-2003, 2004, 2005, 2007, 2008 and 2009, Positive with SHS-2006, 2010 and 2011.

30. DNase (Bacto DNase Test Agar): Negative in common.

31. Phenylalanine deaminase (Phenylalanine/Malonic acid medium, available from Nissui Pharmaceutical Co. Ltd.):

Strains SHS-2003, 2008, 2009, 2010 and 2011: Negative or very weakly positive.

Strains SHS-2004, 2005, 2006 and 2007: Negative.

32. KCN inhibition: Positive in common.

33. Growth in 5% NaCl bouillon: Positive in common.

34. Auxotrophy (Gray & Tatum's medium): Requires nicotinic acid or nicontinamide in common.

35. Ulilization of some organic compouns (D.W. Dye, N.Z. J. Sci., 11, 590-607 (1968), OY medium, 28° C., 20 & 44 hrs, shaking):

    __________________________________________________________________________     Strains                                                                            Ace-   For-                                                                              Fumar-                                                                             Gluco-  Succi-    Galatur-       Propio-                                                                            Tartar-                 SHS-                                                                               tate                                                                              Citrate                                                                            mate                                                                              ate nate                                                                               Malate                                                                             nate                                                                               Benzoate                                                                             onate                                                                               Malonate                                                                            Oxalate                                                                             nate                                                                               ate Lactate             __________________________________________________________________________     2003                                                                               +  +   +  +   +   +   +   -     -    -    -    -   -   +                   2004                                                                               ±                                                                              +   +  +   +   +   +   -     -    -    -    -   -   ±                2005                                                                               ±                                                                              +   +  +   +   +   +   -     -    -    -    -   -   ±                2006                                                                               ±                                                                              +   +  +   +   +   +   -     -    -    -    -   -   ±                2007                                                                               ±                                                                              +   +  +   +   +   +   -     -    -    -    -   -   ±                2008                                                                               +  +   +  +   +   +   +   -     -    -    -    -   -   +                   2009                                                                               +  +   +  +   +   +   +   -     -    -    -    -   -   +                   2010                                                                               +  +   +  +   +   +   +   -     -    -    -    -   -   +                   2011                                                                               +  +   +  +   +   +   +   -     -    -    -    -   -   +                   __________________________________________________________________________      Note:                                                                          (i) Utilizes as a single carbon source (+)                                     (ii) Slightly utilizes or does not utilize as a single carbon source           (±)                                                                         (iii) Does not utilize (-)                                               

36. Ubiquinone *Y. Yamada, K. Aida & T. Uemura, J. Gen. Appl. Microbiol., 15, 181-196 (1969)): strains SHS-2003, 2008: Ubiquinone-8 (and Ubiquinone-7) Strains SHS-2004, 2005, 2006, 2007, 2009 2010 and 2011: Ubiquinone-8.

D. Origin

Strains SHS-2003, 2004, 2005, 2007, 2010 and 2011: Mandarin orange.

Strains SHS-2005 and 2009: Persimmon.

Strains SHS-2008: Soil.

The strains listed in Table 1 were determined by comparing the above-mentioned taxonomical properties of the respective strains in Table 2, with the descriptions in the Bergey's Manual of Determinative Bacteriology 8th Ed. 1974 (hereinafter, will be simply referred to as "Manual") to lead the conclusion which will be described below.

1) Allocation in terms of Family

On the basis of the above described results of the observation that all of the listed strains are short rods of gram-negative and facultatively anaerobic, which form no spores and show a catalase activity but no oxidase activity, they are determined to belong to Family Enterobacteriaceae.

2) Allocation in terms of Genus

All of the listed strains are determined to belong to Genus Erwinia on the basis of the taxonomical properties, particularly on those of, the productions of acids from β-methylglucoside and sucrose (except for SHS-2003 which does not produce acid from sucrose but utilizes the same as a sole carbon source) but not from adonitol, dulcitol or melezitose; no utilization of benzoate, oxalate and propionate; being incapable of hydrolysing starch; being incapable of decarboxylating glutamic acid, arginine, lysine and ortinine (except for SHS-2004, 2005, 2006, and 2007 which decarboxylate glutamic acid) and showing no urease or lipase activity.

3) Allocation in term of species

Strain SHS-2003

Although this strain is considered to be closely related to Erwinia stewartii of group herbicola defined in the Manual on the basis of no flagellation, it still has other taxonomical properties greatly different from those of Erwinia stewartii in the following points:

(i) Requirement of nicotinic acid or nicotinamide for growth,

(ii) Production of hydrogen sulfide from cysteine,

(iii) Reduction of nitrate to nitrite,

(iv) No acid production from sucrose*, arabinose, raffinose or sorbitol,

(v) Production of acids from salicin, cellobiose and glycerol, and

(vi) No utilization of tartarate as a sole carbon source.

Furthermore, since this strain did not show a coincidence with any other known species in the genus defined in the Manual, it was concluded that it should be allocated to a new species, named as Erwinia citreus by the present inventors.

Strains SHS-2004, 2005, 2006 and 2007

Although these strains are likewise considered to be closely related to Erwinia stewartii in view of no flagellation, they still share other common taxonomical properties greatly different from those of Erwinia stewartii in the following points:

(i) Requirement of nicotinic acid of nicotinamide for growth.

(ii) Production of hydrogen sulfide from cysteine,

(iii) Reduction of nitrate to nitrite,

(iv) Decarboxylation of gultamic acid,

(v) No acid production from arabinose, mannitol, lactose or sorbitol,

(vi) Production of acids from salicin and cellobiose, and

(vii) No utilization of tartarate as a sole carbon source.

Furthermore, these strains are observed to be different from the previously described strain SHS-2003 in the points; decarboxylation of glutamic acid; being scarcely capable of utilizing lactose, acetate and lactate as sole carbon source; being unable to produce acids from mannitol and lactose; and having no activity on litmus milk.

Moreover, since these strains did not show coincidences with any other known species in the genus defined in the Manual, it was concluded that they should be allocated to a new species, named as Erwinia punctata by the present inventors.

Of these strains, the strains SHS-2004 and 2006 are common in their taxonomical properties in the points;

(i) No acid production from raffinose, and

(ii) No utilization of mannitol, acetate or lactate.

However, SHS-2004 differs from SHS-2006 in the points:

(i) Production of acetoin from glucose,

(ii) Utilization of nitrate as nitrogen source,

(iii) Production of acid from glycerol, and

(iv) Utilization of formate as a sole carbon source.

On the other hand, the strain SHS-2005 has the same taxonomical properties as those of SHS-2006 in the point of being scarcely capable of utilizing mannitol, acetate and lactate as their sole carbon source, but the former differs from the latter in the points:

(i) Production of acid from glycerol,

(ii) Production of acetoin from glucose,

(iii) Formation of fluorescent pigments in King B medium,

(iv) Scarce production of acid from raffinoise, and

(v) Utilization of formate as a sole carbon source.

Furthermore, the strain SHS-2007 likewise has the properties as those of SHS-2006 in the points of being scarcely capable of utilizing mannitol, acetate and lactate as their sole carbon source, but the former differs from the latter in the pints:

(i) Production of acid from reffinoise,

(ii) Production of acetoin from glucose,

(iii) Utilization of formate as a sole carbon source, and

(iv) Wide temperature range for growth such as 4.0°-47.5° C..

On the basis of the above indicated results of observation, all of the strains SHS-2004, 2005, 2006 and 2007 are likewise found to belong to said Erwinia punctata and each forms a variant with respect to the others.

Strains SHS-2008 and 2010

Although these strains are considered to be closely related to Erwinia tracheiphila or Erwinia quercina in view of their motility with mono-lateral flagellum, the taxonomical properties of them are still greatly different from those of Erwinia tracheiphila defined in the Manual, in the following points:

(i) Growth at a temperature higher than 36° C.,

(ii) Aubundant growth on bouillon agar media,

(iii) Showing mucoid growth,

(iv) Reduction of nitrate to nitrite,

(v) Production of acids from salicin, xylose, melibiose and mannose, and

(vi) Utilization of lactate as a sole carbon source.

On the other hand, there are differences in their taxonomical properties from those of Erwinia quercina defined in the Manual in the following points:

(i) Oxidation of gluconic acid,

(ii) Reduction of nitrate to nitrite,

(iii) Production of acids from melibiose and cellobiose,

(iv) No acid production from mannitol, α-methylglucoside, esucline and sorbitol, and

(v) No gas production on glucose-peptone media.

Moreover, since these strains do not show coincidences with any other known species in the genus defined in the Manual, they were found to be suitably allocated to a new species, named as Erwinia terreus by the present inventors.

Furthermore, the strain SHS-2010 shows substantial coincidence with SHS-2008, except for certain differences observed in the following points:

(i) No acid production from glycerol,

(ii) Production of acid from ribose by SHS-2008 in contrast to very scarce production by SHS-2010,

(iii) Slightly positive in methyl red test, and

(iv) Formation of fluorescent pigments on King B medium.

Strain SHS-2009

This strain is considered to be closely related to Erwinia tracheiphila, E. quercina and E. herbicola, var. herbicola in view of motility with mono-lateral flagellum.

However, there is remarkable differences in the taxonomical properties of this strain as compared with those of Erwinia tracheiphila defined in the Manual in the following points:

(i) Abundant growth in bouillon agar media,

(ii) Growth at a temperature higher than 36° C.

(iii) Showing mucoid growth.

(iv) Reduction of nitrate to nitrite, and

(v) Production of acids from salicin, xylose, melibiose cellobiose, glycerol and mannose.

In addition to this, there is also found remarkable differences from those of Erwinia quercina, in the following points:

(i) Oxidation of gluconate,

(ii) Reduction of nitrate to nitrite,

(iii) Weak formation of acetoin from glucose,

(iv) Production of acids from xylose, melibiose and cellobiose,

(v) No acid production from mannitol, α-methylglucoside, esuclin, ribose or sorbitol,

(vi) No utilization of tartarate as a sole carbon source, and

(vii) No gas production from glucose-peptone medium.

Furthermore, there is still found remarkable difference in the properties of SHS-2010 from those of Erwinia herbicola. var. herbicola in the following points:

(i) Requirement of nicotinic acid or nicotinamide for the growth,

(ii) Weak formation of acetoin from glucose,

(iii) No liquefaction of gelatin,

(iv) Production of acids from melibiose, cellobiose and glycerol, and

(v) No acid production from arabinoise, mannitol, maltose, dextrine, rhamnose, ribose or sorbitol.

Although some differences are still remaining in comparing this strain with the aforedefined SHS-2008, in the points:

(i) Weak acetoin formation from glucose, and

(ii) No acid production from ribose, the former coincides with the latter in the other dominating properties and thus identified to be a variant of the aforedefined Erwinia terreus.

Strain SHS-2011:

This strain is considered to be closely related to Erwinia tracheiphila or Erwinia amylovora in view of its motility with a mono-lateral flagellum.

When the properties of this strain are first compared with those of Erwinia tracheiphila defined in the Manual, there is recognized remarkable differences in the following points;

(i) Moderate growth in bouillon agar media,

(ii) Growth at a temperature higher than 36° C.

(iii) Showing mucoid growth,

(iv) Reduction of nitrate to nitrite,

(v) Production of acids from salicin, xylose, melibiose, cellobiose and mannose, and

(vi) Utilization of lactate as a sole carbon source.

When they are compared with those of Erwinia amylovola, there is recognized remarkable differences in the following points:

(i) Formation of hydrogen sulfide from cysteine,

(ii) Growth at a temperature as high as 36° C., or higher,

(iii) Reduction of nitrate to nitrate,

(iv) No liquefaction of gelatin,

(v) Production of acids from salicin, xylose, melibiose, cellobiose and mannose, and

(vi) No acid production from ribose.

On the other hand, when this strain is compared with the SHS-2008, it is recognized to be in a coincidence with the latter in the properties except for no production of acid from ribose or glycerol, and is identified as a variant of the aforedefined Erwinia terreus.

In addition to those isolated microorganisms (wild strains), any spontaneous mutants obtained therefrom may likewise be utilized in the method of this invention with advantages as far as they are capable of producing 2,5-diketo-D-gluconic acid, and it is needless to say that any strains, obtained by artifically or inductively mutating or modifying these isolated microorganisms so that they exhibit desired properties, may likewise be utilized in embodying the present invention in a smooth way by suitably taming the strains.

The aforedefined microorganisms grow abundantly in an aqueous nutrient medium containing D-glucose, as a main carbon source, corn steep liquor as a nitrogen source and a small amount of inorganic salts. If they are aerobically cultured, they grow on media of very-high-D-glucose concentration with sufficient stability to produce 2,5-diketo-D-gluconic acid (2.5 DKG) in good yeild, as compared with the known microorganisms which have been used in producing the same product.

The concentration of D-glucose in the broth may be as high as 40 w/v % if particularly desired so, though it is usually convenient for an economical production of 2,5-diketo-D-gluconic acid to maintain this concentration to a range of 15-25 w/v % and more preferably, to approximately 20 w/v %.

The temperature of fermentation broth may be in a range of 15-35° C., preferably, in that of 20°-30° C. and more preferably at approximately 28° C. An initial pH value of the medium may be in a range of 5.5-7.5 and preferably in that of 6.0-7.0.

The pH value of the broth may be maintained at a desired range of 4.0-5.5 during the fermentation by incorporating suitable inorganic salts having a buffering action in the starting broth, or by consecutively feeding suitable bases into the broth with the progress of the fermentation. The salt can be exemplified by calcium carbonate and the base can be exemplified by sodium hydroxide.

The inoculated fermentation broth is stirred constantly with an agitator (Ca. 1,740 r.p.m.) under aeration at a ratio of about 600 N ml/min.,

The fermentation completes when the conversion of D-glucose into 2,5 DKG reaches a value which corresponds to about 90 % yield of the latter, which time is about 17-31 hours after beginning cultivation.

The accumulated 2,5 DKG or its salts may be isolated as crystals from the fermentation broth after being treated with any means such as pH adjustment, or alternatively, the broth may advantageously be utilized as such for the nutrient medium of the next stage. For instance, the broth containing 2,5 DKG may directly be utilized for the production of 2-keto-L-gluonic acid, thus the latter can be obtained in high yield in a simple procedure as compared with the conventional method.

In addition to the previously described fermentation, the present invention may be embodied by simply contacting any processed products of the microorganisms with any substrates containing D-glucose. In these cases, conditions similar to those described above in connection with the cultivation of viable cells may also be applied to the incubation of the processed products in the substrate, and the conversion starts immediately after the starting of contact.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following description, the present invention will be elucidated in more detail by way of examples

EXAMPLE 1

(1) Seed medium:

An aqueous solution containing;

    ______________________________________                                         D-glucose (hydrated, content 91%)                                                                       1.0 w/v %,                                            Corn Steep Liquor (CSL)  5.0 w/v %,                                            Potassium bihydrogen phosphate (KH.sub.2 PO.sub.4)                                                      0.1 w/v %,                                            Magnesium sulfate (MgSO.sub.4.7H.sub.2 O)                                                              0.02 w/v %, and                                        Calcium carbonate (CaCO.sub.3)                                                                          0.5 w/v %                                             ______________________________________                                    

is adjusted to pH 6.8-7.0 with 10 % aqueous solution of NaOH, divided into 50 ml portions and placed in sterilized 500 ml conical flasks to give the seed media.

(2) Seed culture:

Each of said media in the flasks is inoculated with one loopful of the strains listed in Table 3 below and shaken (stroke: 71 mm, 270 s.p.m.) at 28° C. for about 8 hours. The culture is terminated when the optical density (O.D.) of the media becomes approximately 8 (end point.

(3) Fermentation broth:

An aqueous solution containing;

    ______________________________________                                         D-glucose          20.0 w/v %,                                                 CSL                 3.0 w/v %,                                                 KH.sub.2 PO.sub.4   0.1 w/v % and                                              CaCO.sub.3          6.3 w/v %                                                  ______________________________________                                    

is adjusted to pH 6.8-7.0, divided into 455 ml portions and placed in 1 L fermentors to give the fermentation broth, into which each 45 ml of said seed media is added.

(4) Fermentation:

The fermentation is carried out under the conditions of:

    ______________________________________                                         Temperature:       28° C.                                               Stirring:          1740 r.p.m.                                                 Aeration:          600 N ml/minute                                             Time:              17-31 hours                                                 ______________________________________                                    

(5) Determination:

The product is chromatographed on a filter paper carrier by ascending under the conditions specified below, and the spots are quantitatively determined by densitometry.

(i) Carrier: Toyo Roshi No. 50

(ii) Developing solvent:

Phenol: Formic acid: Water=75:4:25

(iii) Staining:

Spraying of AHF solution (a solution of water-saturated n-butanol (100 ml) containing anilin (0.93 g) and phthalic acid (1.66 g) followed by a color-developing treatment at 105° C. for 2 minutes.

(vi) Color and Rf value:

    ______________________________________                                         Substance           Color:  Rf:                                                ______________________________________                                         2,5-diketo-D-gluconic acid                                                                         Brown   0.16-0.18                                          2-keto-D-gluconic acid                                                                             Pink    0.27-0.29                                          D-glucose           Brown   0.48-0.50                                          ______________________________________                                    

In addition to this, a thin layer chromatography is also performed with "TLC alumisheet cellulose (tradename of E Merck A.G.) and the same developing solvent system and staining operation is described above. In this case, the determination is performed by comparison of the chromatogram with that obtained with an authentic specimen.

(6) Termination:

The fermentation is terminated at a time when the pink spot of 2-keto-D-gluconic acid disappears from said paper or thin layer chromatogram.

(7) Results:

                  TABLE 3                                                          ______________________________________                                                      Fermentation Culture                                                                          2,5-diketo-D-                                      Seed Culture                gluconic acid.                                     At termination At termination                                                                              Conc.   Yield for                                  Strains            Time           Time  (w/v  D-Glu-                           SHS-  pH    OD     (hrs.)                                                                               pH  OD   (hrs.)                                                                               %)    cose (%)                         ______________________________________                                         2003  7.0   10.0   8     5.4 12.3 18    19    88                               2004  6.0   8.5    8     4.9 12.0 17    19    89                               2005  6.9   9.5    9     5.1 12.6 20    19    86                               2006  5.9   7.5    8     4.7 12.0 21    19    89                               2007  6.0   7.0    8     4.9 12.6 17    19    89                               2008  5.6   3.1    10    5.3 12.4 29    17    70                               2009  5.8   3.4    10    5.7 12.4 21    18    79                               2010  5.4   2.5    10    5.4 12.8 31    18    75                               2011  5.3   2.4    10    5.1 12.2 31    17    75                               ______________________________________                                    

EXAMPLES 2 AND 3 Productions with cell suspension and with crude enzyme extract

(1) Medium used for obtaining microorganism cells:

An aqueous solution containing: Yeast extract (available from Daigo Eiyo Yakuhin K.K.)

    ______________________________________                                         Yeast extract (available from Daigo Eiyo                                                                 0.1 w/v %                                            Kauhin K.K.)                                                                   KH.sub.2 PO.sub.4         0.1 w/v %                                            NaSO.sub.4.7H.sub.2 O    0.02 w/v % and                                        CaCO.sub.3                0.6 w/v %                                            ______________________________________                                    

is adjusted to pH 7.0, divided into 80 ml portions, and placed in conical flasks (500 ml).

(2) Cultures for collecting microorganism cells and for separating crude enzyme extract:

Each of said media is inoculated with the strains listed in Table 4 below in a similar manner as described in the seed culture of Example 1 and shaken (stroke, 71 mm, 270 s.p.m.) at 28° C. for 16 hours.

After the shaking, the microorganism cells are collected by centrifuging the cultured media, washed twice with saline and divided into two portions.

One of said portions is suspended again in saline to give the cell suspension. The other is suspended in a 1/50 M tris-HC1 buffer (pH 7.5) wherein the cells are disruptured by ultra-sonic treatment and centrifuged to remove insoluble residue therefrom in order to obtain crude cell-free enzyme extract as its supernatant.

(3) Incubation with cell suspension:

A mixture is prepared with 1/10 M 3,3-dimethylgultaric acid buffer (pH 5.0) containing about 5 w/v % of D-glucose, wherein the cell concentration is adjusted so that the O.D. at 660 mμ is approximately 10. The mixture is divided into 10 ml portions, placed in test tubes of 23 mm (diameter) x 196 mm (length) and incubated at 28° C. for 3 hours.

The mixture is centrifuged to give a supernatant which is analyzed by paper chromatography. The results are summarized in Table 4 below, wherein the concentrations of D-glucose in the mixture before incubation are also given.

                  TABLE 4                                                          ______________________________________                                         (Concentrations after 3 hours shaking)                                         D-glucose (%)                                                                        before                                                                   Strains                                                                              incuba-         2-keto-D-gluconic                                                                         2,5-diketo-D-gluco-                           SHS-  tion            acid (%)   nic acid (%)                                  ______________________________________                                         2003  4.9      2.7    0.8        1.9                                           2004  5.0      1.9    0.7        1.5                                           2005  5.0      2.6    0.6        0.7                                           2006  5.0      1.9    0.9        1.1                                           2007  5.0      1.4    0.3        1.4                                           2008  5.0      1.3    0.8        1.2                                           2009  5.2      1.9    0.7        1.3                                           2010  5.0      2.9    0.5        0.9                                           2011  5.0      2.8    0.7        0.9                                           ______________________________________                                    

(4) Incubation with crude enzyme extract:

To a mixture prepared in a similar manner as described in (3) above, the crude enzyme extract is added so that its concentration is 0.25 mg/ml, determined as a protein quantity (Folin's method). After being shaken as previously described, the mixture is treated with two drops of 10 w/v % trichloroacetic acid solution to remove the protein portion, and chromatographed on a filter paper. The results are shown in Table 5 below.

                  TABLE 5                                                          ______________________________________                                         (Concentrations after 3 hours shaking)                                         Strain                  2,5-diketo-D-gluconic acid                             SHS-  2-keto-D-gluconic acid (%)                                                                       (%)                                                    ______________________________________                                         2003  1.0               1.0                                                    2004  0.6               0.8                                                    2005  0.4               0.4                                                    2006  0.5               0.5                                                    2007  0.3               0.4                                                    2008  0.8               0.5                                                    2009  0.4               0.6                                                    2010  0.3               0.3                                                    2011  0.3               0.3                                                    ______________________________________                                     

What is claimed is:
 1. A method for preparing 2,5-diketo-D-gluconic acid or a salt thereof which comprises converting D-glucose into 2,5-diketo-D-gluconic acid in the presence of a strain of microorganisms consisting essentially of Erwinia citreus which is non-motile and does not effectively grow at a temperature above 39° C., and produces a recoverable quantity of 2,5-diketo-D-gluconic acid or a salt thereof by cultivation in the presence of D-glucose and utilizing formate, but is incapable of liquefying gelatin and of producing any acid from esculin, rhamnose or sorbitol, respectively, or growth and isolating the 2,5-diketo-D-gluconic acid.
 2. A method for preparing 2,5-diketo-D-gluconic acid or a salt thereof which comprises converting D-glucose into 2,5-diketo-D-gluconic acid in the presence of a strain of microorganisms consisting essentially of Erwinia punctata which is non-motile and does not effectively grow at a temperature above 39° C., and produce a recoverable quantity of 2,5-diketo-D-gluconic acid or a salt thereof by cultivation in the presence of D-glucose, and forming reducing compound from sucrose, but is incapable of liquefying gelatin and of producing any acid from mannitol, lactose, esculin, glycerol, rhamnose or sorbitol, respectively, on growth and isolating the 2,5-diketo-D-gluconic acid.
 3. A method for preparing 2,5-diketo-D-gluconic acid or a salt thereof which comprises converting D-glucose into 2,5-diketo-D-gluconic acid in the presence of a strain of microorganisms consisting essentially of Erwinia terreus which does not effectively grow at a temperature above 40° C., and producing a recoverable quantity of 2,5-diketo-D-gluconic acid or a salt thereof by cultivation in the presence of D-glucose, forming reducing compound from sucrose and acetoin, and utilizing formate, but is incapable of liquefying geletin and of producing any acid from mannitol, esculin, rhamnose or sorbitol, respectively, on growth and isolating the 2,5-diketo-D-gluconic acid.
 4. A method for preparing 2,5-diketo-D-gluconic acid or a salt thereof which comprises converting D-glucose into 3,5-diketo-D-gluconic acid in the presence of at least one strain of 2,5-diketo-D-gluconic acid producing microorganisms selected from the group consisting of Erwinia citreus, Erwinia punctata and Erwinia terreus which does not effectively grow at a temperature above 47.5° C. and producing a recoverable quantity of 2,5-diketo-D-gluconic acid or salt thereof by cultivation in the presence of D-glucose, but is incapable of liquiefying geletin and does not produce gas or any acid from esculin, rhamnose or sorbitol, respectively, on growth and isolating the 2,5-diketo-D-gluconic acid.
 5. The method of claim 26, wherein at least one strain of said microorganisms is cultivated in an aqueous nutrient medium in the presence of D-glucose to produce and accumulate 2,5-diketo-D-gluconic acid or any salts thereof in the medium during the cultivation. 